Ribonucleases, in particular, Rap (Lee, Exp Opin Biol Ther 2008; 8:813-27) and its more basic variant, amphinase (Ardelt et al., Curr Pharm Biotechnol 2008:9:215-25), are potential anti-tumor agents (Lee and Raines, Biodrugs 2008; 22:53-8). Rap is a single-chain ribonuclease of 104 amino acids originally isolated from the oocytes of Rana pipiens. Rap exhibits cytostatic and cytotoxic effects on a variety of tumor cell lines in vitro, as well as antitumor activity in vivo. The amphibian ribonuclease enters cells via receptor-mediated endocytosis and once internalized into the cytosol, selectively degrades tRNA, resulting in inhibition of protein synthesis and induction of apoptosis.
Rap has completed a randomized Phase Mb clinical trial, which compared the effectiveness of Rap plus doxorubicin with that of doxorubicin alone in patients with unresectable malignant mesothelioma, with the interim analysis showing that the MST for the combination was 12 months, while that of the monotherapy was 10 months (Mutti and Gaudino, Oncol Rev 2008; 2:61-5). Rap can be administered repeatedly to patients without an untoward immune response, with reversible renal toxicity reported to be dose-limiting (Mikulski et al., J Clin Oncol 2002; 20:274-81; Int J Oncol 1993; 3:57-64).
Rap and other toxins or cytotoxins may be conjugated to antibodies or antibody fragments for targeted delivery to selected disease-associated cells, such as cancer cells or autoimmune disease cells. An exemplary tumor-associated antigen is EGP-1, also known as Trop-2.
Trop-2 is a type-I transmembrane protein and has been cloned from both human (Fornaro et al., Int J Cancer 1995; 62:610-8) and mouse cells (Sewedy et al., Int J Cancer 1998; 75:324-30). In addition to its role as a tumor-associated calcium signal transducer (Ripani et al., Int J Cancer 1998; 76:671-6), the expression of human Trop-2 was shown to be necessary for tumorigenesis and invasiveness of colon cancer cells, which could be effectively reduced with a polyclonal antibody against the extracellular domain of Trop-2 (Wang et al., Mol Cancer Ther 2008; 7:280-5).
The growing interest in Trop-2 as a therapeutic target for solid cancers (Cubas et al., Biochim Biophys Acta 2009; 1796:309-14) is attested by further reports that documented the clinical significance of overexpressed Trop-2 in breast (Huang et al., Clin Cancer Res 2005; 11:4357-64), colorectal (Ohmachi et al., Clin Cancer Res 2006; 12:3057-63; Fang et al., Int J Colorectal Dis 2009; 24:875-84), and oral squamous cell (Fong et al., Modern Pathol 2008; 21:186-91) carcinomas. The latest evidence that prostate basal cells expressing high levels of Trop-2 are enriched for in vitro and in vivo stem-like activity is particularly noteworthy (Goldstein et al., Proc Natl Acad Sci USA 2008; 105:20882-7).
The murine anti-Trop-2 mAb, mRS7, was generated by hybridoma technology using a crude membrane preparation derived from a surgically removed human primary squamous cell carcinoma of the lung as immunogen (Stein et al., Cancer Res 1990; 50:1330-6). Immunoperoxidase staining of frozen tissue sections indicated that the antigen defined by mRS7 is present in tumors of the lung, stomach, bladder, breast, ovary, uterus, and prostate, with most normal human tissues being unreactive (Stein et al., Int J Cancer 1993; 55:938-46). The antigen recognized by mRS7 was later shown to be a 46-48 kDa glycoprotein and named epithelial glycoprotein-1, or EGP-1 (Stein et al., Int J Cancer 1994; 8:98-102), which is also referred to in the literature as Trop-2 (Ripani et al., Int J Cancer 1998; 76:671-6). Upon binding to the target cells, mRS7 is rapidly internalized within 2 h (Stein et al., Int J Cancer 1993; 55:938-46).
Radiolabeled mRS7 has been shown to effectively target and treat cancer xenografts in nude mice in several earlier studies (Stein et al., Antibody Immunoconj Radiopharm 1991; 4:703-12; Stein et al., Cancer 1994; 73:816-23; Shih et al., Cancer Res 1995; 55:5857s-63s; Stein et al., J Nucl Med 2001; 42:967-74; Stein et al., Crit Rev Oncol Hematol 2001; 39:173-80). However, a need exists in the field for immunoconjugates (“immunotoxins”) of RS7 or other disease-targeting antibodies that may be attached to Rap or other cytotoxins to provide a more efficacious agent for disease therapy.